Selenium modification of tRNAs has been previously demonstrated in broken cell preparations from Methanococcus vannielii. Chromatography of the extracts from these bacteria on phenyl Sepharose yielded active enzyme(s). Added M. vannielii bulk tRNA was used as substrate. Consistent with the results obtained from the broken cell preparations, ATP and o-acetyl serine were required for full activity. Dilution of 75Se incorporated into the tRNA by unlabeled L-selenocysteine suggests that this amino acid may serve as the selenium donor. Nucleoside analysis of the labeled tRNA mixtures revealed two unidentified radioactive peaks on the HPLC profile in addition to 5-methylaminomethyl-2-selenouridine. Initial characterization of a second as yet unidentified naturally occurring selenonucleoside from M. vannielii was also performed.